Niemann-Pick C1-Like 1, NPC1L1, is crucial for cholesterol absorption and is the molecular target for ezetimibe, the cholesterol lowering drug. Previous studies showed that NPC1L1 expression is increased in patients with diabetes mellitus contributing to associated hypercholesterolemia. Blocking choelsterol absoprtion with ezetimibe in combination with statins (inhibitors of cholesterol synthesis) has been shown to be more effective than treatment with statins alone in lowering serum cholesterol. Current guidelines recommend aggressive lowering of blood cholesterol in patients with high risk for developing cardiovascular diseases (CVD), such as diabetic patients. Achieving these stringent low targets remains challenging. Since ezetimibe only blocks NPC1L1 activity, decreasing NPC1L1 expression represents an attractive therapeutic approach for further reduction in plasma cholesterol. Therefore, it is critical to delineate cellular pathways involved in decreasing NPC1L1 expression that could be exploited to efficiently reduce the risk for CVD. We have previously shown that NPC1L1 expression is regulated by an epigenetic mechanism involving DNA methylation. However, the role of epigenetic histone modifications in modulating NPC1L1 expression is not known. Our preliminary data showed that the HDAC inhibitors (HDACi) butyrate and valproic acid (VPA) significantly decreased NPC1L1 expression in intestinal Caco2 cells and mouse intestine. Also, siRNA-mediated attenuation of HDAC2 and 3, but not the other isoforms, reduced NPC1L1 expression; however, the underlying molecular mechanisms remain unclear. We hypothesize that specific histone deacetylase isoforms modulate NPC1L1 expression via modifying chromatin structure and/or altering the binding of specific transcription factors to the NPC1L1 gene. We also hypothesize that HDAC inhibitors such as VPA decrease cholesterol absorption and reduce plasma cholesterol in mouse models of hypercholesterolemia. VPA is a widely used drug for the treatment of epilepsy and bipolar disorders. Therefore, it will be important to directly study its effects on cholesterol absorption in humans. However, the current standard method to directly measure cholesterol absorption has limitations in humans as it relies on the use of isotope-labeled cholesterol and laborious laboratory techniques. We are currently inveatigating a novel simple assay that is non-radioactive to evaluate cholesterol transport. Our preliminary data indicate that this method is suitable for assessing NPC1L1 function. We hypothesize that our putative simple non-radioactive approach to measure NPC1L1 function represents a novel method to directly measure cholesterol absorption in living animals. The studies are designed to rigorously examine the hypotheses and address two main objectives. In Specific Aim 1, our studies are aimed to investigate the effects of attenuating HDAC isoforms on NPC1L1 function, to examine chromatin remodeling of NPC1L1 gene and alterations in binding of transcription factors in intestinal cell lines and enteroids, and to investigate the effects of HDACi on cholesterol absorption in the LDL receptor knockout mice as a model of hypercholesterolemia. Studies proposed in Specific Aim 2 are designed to establish the accuracy of the putative method in measuring cholesterol absorption utilizing NPC1L1 knockout mice and wild type littermates. The studies will also investigate the decrease in cholesterol absorption by HDACi using the new method in mice, providing the basis for future translational studies in humans. Notably, the risk for CVD is significantly higher in the veterans as compared to the general population. Thus, unraveling novel mechanisms involving histone deacetylation to downregulate NPC1L1 expression and establishing novel, state-of-the-art method to measure the associated decrease in cholesterol absorption are timely and directly relevant to improving the health of the veterans.